**2.1. Tissue collection from Galapagos sea lion pups**

In a recent study [35], blubber biopsy and hair samples of 20 Galapagos sea lion pups of 2– 12 months of age were obtained from four rookeries in the Galapagos Archipelago (3°N−4°S, 87°−94°W) between 24-29March 2008. Briefly, pups were sampled at Isabela (Loberia Chica, *n* = 5), Floreana, (Loberia, *n* =6) and Santa Cristobal (Puerto Baquerizo, *n* = 4; Isla Lobos, *n* = 5) islands. Pups were captured with hoop nets and manually restrained. Age was estimated by visual observation of both the size and weight of the animal. In all circumstances, capture stress and holding time were minimized (< 10-15 min). Hair samples were obtained using a sterile scissor to trim or a scalpel to shave the region to be used prior to the biopsy collection and deposited into labelled zipper bags. Biopsies (100 mg; 6mm−Miltex biopsy punch) were collected from an area 10-20 cm lateral to the spinal column and anterior to the pelvis. The biopsy site was pre-cleaned with alcohol and betadine. Biopsies were wrapped in hexanerinsed aluminum foil and placed in a cooler with wet ice and transferred into cryovals placed in a cryoship (-20°C) during the field sampling, and, afterwards stored at -80C in the laboratory until chemical analysis.

Pups were chosen because (a) the animals are readily accessible and relatively easy to capture in most of the rookeries of the Galapagos Islands year round; (b) the animals are of similar age (3-10 months), minimizing the influence of life history parameters on contaminant concentrations; (c) because they are nursed by adult reproductive females they have a high trophic position because they are feeding on mother's milk, ingesting energy and pollutants and analogous to a predator–prey relationship [35]. The rationale of the

study design to justify the use of pups as ecosystem based sentinels of biomagnification is also explained in Figure 1.

Assessing Biomagnification and Trophic Transport of Persistent Organic Pollutants in the Food Chain of the Galapagos Sea Lion (*Zalophus wollebaeki*): Conservation and Management Implications 81

March-April 2008. Mullets are coastal fish, inhabiting nearshore habitats, and demersalbenthic feeders (detritivorous), grazing on detritus and bottom sediments and digesting the nutritive matter (iliophagous foraging), while Galapagos thread herrings are endemic, pelagic and schooling fishes that filter-feed (planktivorous) mainly on tiny planktonic

After field collection, fish specimens were frozen until further transportation to the lab, where they were stored at -80ºC. Each fish was measured, weighed and sexed. Muscle biopsies were extracted from the dorsal, lateral muscle of each fish, using a 6mm–biopsy

Each individual fish was homogenized using a clean, hexane-acetone rinsed meat grinder (Omcam Inc., Italy). The ground fish was then further homogenized in a homogenizer (Omni, USA and/or Polytron, Kinematica, GmbH, Switzerland) at dial position 5-6 for ≈1 min until material was well mixed and homogenous in appearance. Homogenized samples and subsamples were transferred to clean glass jars and stored at -80 ºC until further

Each set of hair samples collected from Galapagos sea lion pups was cleaned for lipid and particle removal by washing the hair three times with a chloroform:methanol 2:1 v/v solution using a clean Pasteur glass pipette. Samples were transferred into labelled scintillation vials and desiccated overnight, and, then, lyophilized using a freeze drier (Free Zone ® Plus 4.5 Liter Cascade; Labconco, Kansas City, MO) for 24 hr (Vacuum pressure set

Fish biopsy samples were freeze dried overnight (Vacuum pressure set point: 0.01 mBar). Biopsy samples were weighed and freeze dried again to determine if there were differences in weights after the second freeze drying. Once the sample weight was constant (i.e., no remaining moisture), one set of freeze dried samples was stored in the desiccator until further analysis for *δ*15*N*. The set of freeze dried replicates underwent an extraction protocol to remove lipids to be used for *δ*13*C* analysis. First, freeze dried samples were pulverized using a mortar and transferred into a glass tube for lipid extraction by adding 5ml of chloroform:methanol 2:1 v/v; and, then vortex mixed for 30 seconds. Solids were dispersed with sonification in bath sonicator for 10 min. Samples were allowed to settle for 30 min at room temperature, followed by an additional 30 second vortex and sonification. Samples were centrifuged for 5 minutes at 1000 rpm (model GS6R, Beckman, USA) to enhance pellet formation. The solvent was carefully removed with glass Pasteur pipette (pipette was changed for each sample), without transferring any particulate matter, and the solvent was disposed in the waste bottle. A second extraction was repeated. The supernatant was carefully removed with pipette and the residue was left at -20ºC overnight. Samples were dried under Nitrogen and transferred to a clean, amber vial for analysis of stable isotopes of

organisms (e.g., phytoplankton) in open waters [36].

chemical analysis.

point: 0.01 mBar).

carbon and nitrogen.

punch (Accuderm, USA), and saved in vials for stable isotope analysis.

**2.3. Sample preparation for Stable Isotopes Analysis (SIA)** 

**Figure 1.** Conceptual model illustrating the bioaccumulation process in a representative, food chain of the Galapagos sea lion. Piscivorous Galapagos sea lions can be exposed to persistent organic pollutants (POPs), mainly through dietary ingestion. Low trophic level prey fish can absorb POPs from water and plankton (planktivorous fish), as well as from sediments (detritivorous fish). Nursing pups can bioaccumulate POPs from adult females by nursing and thus occupy a higher tropic level relative to their mothers because*δ*15*N* isotopic enrichment.
